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Chapter 6

Performing the Pure Dye Calibration

Evaluating Pure Dye Calibration Spectra

Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Installation and Maintenance Guide

Notes

7300 System

ROX

~610 nm

SYBR
Green

~520 nm

TAMRA C

~580 nm

VIC B

~550 nm

Table 1

Spectra for pure dyes supported by the 7300 system

(continued)

Instrument Pure

Dye

Filter

Peak

(nm)

Summary of Contents for 7300

Page 1: ...nt Performing the Regions of Interest ROI Calibration Performing the Background Calibration and Optical Calibration Performing the Pure Dye Calibration Verifying the Instrument Performance Powering Off the 7300 7500 7500 Fast System Applied Biosystems 7300 7500 7500 Fast Real Time PCR System Installation and Maintenance Guide ...

Page 2: ...TE QUANTIFICATION OR RELATIVE QUANTIFICATION AND THE APPLIED BIOSYSTEMS 7300 7500 7500 FAST REAL TIME PCR SYSTEMS GETTING STARTED GUIDES FOR ALLELIC DISCRIMINATION AND PLUS MINUS DETECTION FOR LIMITED LABEL LICENSE OR DISCLAIMER INFORMATION TRADEMARKS Applera Applied Biosystems AB Design ABI PRISM and VIC are registered trademarks and FAM JOE NED ROX and TAMRA are trademarks of Applera Corporation...

Page 3: ...on and Maintenance Guide Set up the computer Computer Setup Guide Set up the computer Set up the System Installing the Software and Powering On the Instrument Performing the ROI Calibration Performing the Background Calibration Performing the Pure Dye Calibration Verifying the Instrument Performance Chapter Icon Unpacking and Setting Up the Instrument Before you begin the installation To install t...

Page 4: ...Read This First iv Installation and Maintenance Guide for the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System ...

Page 5: ...aring the TaqMan RNase P Verification Plate Creating a Plate Document for the RNase P Verification Run Analyzing the TaqMan RNase P Plate Data Performing the Verification Run Analyzing the Pure Dye Calibration Data Preparing for the Calibration Preparing Plates and Performing the Pure Dye Calibration Creating a Plate Document for the Pure Dye Calibration Performing the Optical Calibration for the ...

Page 6: ...vi Applied Biosystems 7300 7500 7500 Fast Real Time PCR System Installation and Maintenance Guide DRAFT May 15 2005 10 11 pm Instl_Workflow fm ...

Page 7: ... xviii General Instrument Safety xx Chemical Safety xxi Chemical Waste Safety xxii Electrical Safety xxiii Physical Hazard Safety xxiv Biological Hazard Safety xxiv Workstation Safety xxv Safety and Electromagnetic Compatibility EMC Standards xxv Chapter 1 Before Starting the Installation 1 Setting Up the Computer 2 Selecting the Installation Method 2 Planning the Installation 5 Chapter 2 Unpackin...

Page 8: ...Preparing the Background Calibration Plate 52 Creating a Plate Document for the Background Calibration 54 Performing the Background Calibration 55 Analyzing the Background Calibration Data 56 Performing the Optical Calibration for the 7500 7500 Fast System 59 Troubleshooting 64 Chapter 6 Performing the Pure Dye Calibration 67 Overview 68 Preparing for the Calibration 71 Creating a Plate Document f...

Page 9: ...5 Monitoring Lamp Status 117 Replacing the Halogen Lamp 118 Replacing the Instrument Fuses 122 Updating the Operating System Software and Service Packs 124 Appendix B Creating a Background Plate 125 Overview 125 Creating a Background Plate 125 Appendix C Creating a Custom Pure Dye Plate 127 Overview 127 Determining Optimum Dye Concentration 128 Creating a Custom Pure Dye Plate 130 Adding the Custo...

Page 10: ...x Applied Biosystems 7300 7500 7500 Fast Real Time PCR System Installation and Maintenance Guide ...

Page 11: ...ot their system Assumptions This manual assumes that you Are familiar with the Microsoft Windows XP operating system Understand general techniques for preparing and handling DNA samples Have a general understanding of hard drives and data storage file transfers and copying and pasting Text Conventions This guide uses the following conventions to make text easier to understand Bold indicates user a...

Page 12: ... installation CD Note For additional documentation see How to Obtain Support on page xiii Send Us Your Comments Applied Biosystems welcomes your comments and suggestions for improving its user documents You can e mail your comments to techpubs appliedbiosystems com Document Title Online Help P N P N Applied Biosystems 7300 7500 7500 Fast Real Time PCR System Plus Minus Detection Getting Started Gu...

Page 13: ...lick the link for Support At the Support page you can Obtain worldwide telephone and fax numbers to contact Applied Biosystems Technical Support and Sales facilities Search through frequently asked questions FAQs Submit a question directly to Technical Support Order Applied Biosystems user documents MSDSs certificates of analysis and other related documents Download PDF documents Obtain informatio...

Page 14: ...Preface How to Obtain Support xiv Applied Biosystems 7300 7500 7500 Fast Real Time PCR System Installation and Maintenance Guide ...

Page 15: ...following topics Safety Conventions Used in This Document xvi Symbols on Instruments xvii Safety Labels on Instruments xviii General Instrument Safety xx Chemical Safety xxi Chemical Waste Safety xxii Electrical Safety xxiii Physical Hazard Safety xxiv Biological Hazard Safety xxiv Workstation Safety xxv Safety and Electromagnetic Compatibility EMC Standards xxv ...

Page 16: ...ndicates an imminently hazardous situation that if not avoided will result in death or serious injury This signal word is to be limited to the most extreme situations Except for IMPORTANTs each safety alert word in an Applied Biosystems document appears with an open triangle figure that contains a hazard symbol These hazard symbols are identical to the hazard icons that are affixed to Applied Bios...

Page 17: ... not a protected ground terminal Indicates the Off position of the main power switch Indicates a protective grounding terminal that must be connected to earth ground before any other electrical connections are made to the instrument Indicates a standby switch by which the instrument is switched on to the Standby condition Hazardous voltage may be present if this switch is on standby Indicates a te...

Page 18: ... chimiques dangeureux Lire les fiches techniques de sûreté de matériels avant la manipulation des produits CAUTION Hazardous waste Refer to MSDS s and local regulations for handling and disposal ATTENTION Déchets dangereux Lire les fiches techniques de sûreté de matériels et la régulation locale associées à la manipulation et l élimination des déchets WARNING Hot lamp AVERTISSEMENT Lampe brûlante ...

Page 19: ...s 7300 7500 7500 Fast Real Time PCR System Installation and Maintenance Guide xix Locations of Warnings The Applied Biosystems 7300 7500 7500 Fast Real Time PCR System contain warnings at the locations shown below Attention Physical hazard Physical hazard Attention Attention Attention ...

Page 20: ...assistance of others Depending on the weight of the computer and or the monitor moving them may require two or more people Things to consider before lifting the computer and or the monitor Make sure that you have a secure comfortable grip on the computer or the monitor when lifting Make sure that the path from where the object is to where it is being moved is clear of obstructions Do not lift an o...

Page 21: ... clothing and gloves when handling reagent and waste bottles About MSDSs Chemical manufacturers supply current Material Safety Data Sheets MSDSs with shipments of hazardous chemicals to new customers They also provide MSDSs with the first shipment of a hazardous chemical to a customer after an MSDS has been updated MSDSs provide the safety information you need to store handle transport and dispose...

Page 22: ...Data Sheets and local regulations for handling and disposal CHEMICAL WASTE HAZARD Wastes produced by Applied Biosystems instruments are potentially hazardous and can cause injury illness or death CHEMICAL STORAGE HAZARD Never collect or store waste in a glass container because of the risk of breaking or shattering Reagent and waste bottles can crack and leak Each waste bottle should be secured in ...

Page 23: ...ry Ensure the health and safety of all personnel in your laboratory Ensure that the instrument waste is stored transferred transported and disposed of according to all local state provincial and or national regulations IMPORTANT Radioactive or biohazardous materials may require special handling and disposal limitations may apply Electrical Safety ELECTRICAL SHOCK HAZARD Severe electrical shock can...

Page 24: ...arts PHYSICAL INJURY HAZARD Moving parts can crush and cut Keep hands clear of moving parts while operating the instrument Disconnect power before servicing the instrument Biological Hazard Safety General Biohazard BIOHAZARD Biological samples such as tissues body fluids and blood of humans and other animals have the potential to transmit infectious diseases Follow all applicable local state provi...

Page 25: ...EMC Standard European Safety and EMC Standards Australian EMC Standards U S and Canadian Safety Standards This instrument has been tested to and complies with standard UL 61010A 1 Safety Requirements for Electrical Equipment for Laboratory Use Part 1 General Requirements and with standard UL 61010 2 010 Particular Requirements for Laboratory Equipment for the Heating of Materials This instrument h...

Page 26: ...on and immunity EMC Directive 89 336 EEC This instrument has been tested to and complies with standard EN 61326 Group 1 Class B Electrical Equipment for Measurement Control and Laboratory Use EMC Requirements Australian EMC Standards This instrument has been tested to and complies with standard AS NZS 2064 Limits and Methods Measurement of Electromagnetic Disturbance Characteristics of Industrial ...

Page 27: ...00 7500 7500 Fast System Installing and Setting Up the Software Performing the ROI Calibration Performing the Background Calibration and Optical Calibration Performing the Pure Dye Calibration Verifying the Instrument Performance Installing the Software and Powering On the Instrument See page 5 Planning the Installation See page 2 Setting Up the Computer See page 2 Selecting the Installation Metho...

Page 28: ... installing and calibrating the system Using the Real Time System Setup Wizard Using the Wizard to Install the System The Real Time System Setup wizard is an online tool that you can use instead of this Installation Guide to help you install your new system After you set up the computer and log in as a Windows administrator as described in the Applied Biosystems Real Time PCR System Computer Setup...

Page 29: ...es the level of background fluorescence in the instrument During a run the software removes the background fluorescence from the run data Once a month After replacing the halogen lamp After a background recalibration you must perform an optical 7500 7500 Fast systems only Optical 7500 7500 Fast systems only Compensates for the physical effects of the additional filter present in 7500 7500 Fast ins...

Page 30: ...ide Using This Guide to Recalibrate the System You can recalibrate the system manually after the system is up and running by following the procedures in this guide However the Real Time System Setup wizard automates the recalibrations and eliminates the need for manual inspection of pure dye spectra Chapter 6 and manual calculation of system verification values Chapter 7 Installing the Software an...

Page 31: ... page in the Real Time System Setup wizard for steps and times for the wizard 00 30 00 00 10 00 00 45 00 00 15 00 Unpack and set up the instrument Chapter 2 a Select additional hardware and software b Uncrate and place the 7300 7500 7500 Fast system c Check the shipped materials d Set Up the 7300 7500 7500 Fast System Install and set up the software Chapter 3 a Install the SDS Software for the 730...

Page 32: ...P verification plate b Create a plate document c Perform the verification run d Analyze the TaqMan RNase P plate data 7500 Fast 00 40 00 7500 7500 Fast 7300 Note You must be present at the instrument for approximately 5 minutes of the total run time 7500 Fast Recommended Workflow continued See Note below Perform the optical calibration 7500 7500 Fast systems only Chapter 5 a Create a plate documen...

Page 33: ...oftware and Powering On the Instrument Performing the ROI Calibration Performing the Background Calibration and Optical Calibration Performing the Pure Dye Calibration Verifying the Instrument Performance Before Starting the Installation See page 20 See page 14 See page 12 Setting Up the 7300 7500 7500 Fast Instrument Checking the Shipped Materials Uncrating and Placing the 7300 7500 7500 Fast Sys...

Page 34: ...s Required Getting Started Before you begin installing the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System Set up the computer as described in the Applied Biosystems Real Time PCR System Computer Setup Guide Read the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System Site Preparation Guide P N 4378654 and complete the preinstallation checklist contained in the guide Obtain the...

Page 35: ...y UPS Applied Biosystems recommends the use of a 1 5 kVA uninterruptable power supply UPS especially in areas prone to power failure Power failures and other events that abruptly terminate the function of the 7300 7500 7500 Fast system can corrupt data and possibly damage the system IMPORTANT UPSs have finite battery lives and consequently provide power for a limited time from 30 minutes to severa...

Page 36: ...your system By saving your sds and sdt files to one or more writable CDs on a weekly basis you can effectively back up the data generated by your 7300 7500 7500 Fast system Before installing the 7300 7500 7500 Fast system decide on a method for backing up your data Installing Software to the 7300 7500 7500 Fast System If you want to install additional software to the 7300 7500 7500 Fast system com...

Page 37: ...g system installed on the computer shipped with the 7300 7500 7500 Fast system contains a native firewall utility Applied Biosystems does not support the use of the Windows firewall software and cannot provide support for problems arising through its use System Utilities or Performance Optimizing Software Applied Biosystems recommends the regular use of the Windows XP Professional operating system...

Page 38: ...e PCR System Site Preparation Guide The guide is shipped to you before the instrument arrives and contains important environmental and electrical requirements for the 7300 7500 7500 Fast system Guidelines for Lifting and Moving Verify that the surface you will be placing the instrument on supports at least 54 5 kg 120 lbs Verify that the pathway to the final position of the instrument is clear of ...

Page 39: ...maged contact Applied Biosystems If no items are damaged continue with the installation 2 Cut the straps securing the instrument crate 3 Cut the tape securing the top flaps of the instrument crate and open them 4 Remove the Packing Kit from the instrument and set it aside 5 Lift and remove the lid from the instrument crate 6 Remove the protective cover from the 7300 7500 7500 Fast instrument 7 Pos...

Page 40: ... 3 2 1 8 7 6 5 _ 0 9 Backspace R E W Q I U Y T O P F D S A K J H G L V C X Z M N B Enter Shift Shift Ctrl Alt Caps Lock Tab Page Up Home Insert End Page Down 3 2 1 4 6 5 8 7 9 Enter 0 Alt Ctrl Pause Break Print Screen SysRq Scroll Lock Delete Num Lock Instrument and Computer 7300 7500 7500 Fast instrument Dell Tower Computer Dell Flat Screen Monitor 7300 7500 7500 Fast instrument Dell Laptop Compu...

Page 41: ...te Tool Black Optical Plate Tool Splash Free Support Base Optical Reaction Plates Packing Kit P N 7300 7500 Packing kit 4349804 Primer Express Software Kit 4361938 Universal Voltage Kit Australian British European North American and Japanese Power Cords 603615 Optical Reaction Plates 96 well with bar codes 100 plates 4306737 Universal Serial Bus USB Cable 4328260 Halogen Lamp 12V 75W 4345287 Green...

Page 42: ...cking Kit P N 7500 Fast Packing kit 4361854 Primer Express Software Kit Version 3 0 4361938 Universal Voltage Kit Australian British European North American and Japanese Power Cords 603615 Universal Serial Bus USB Cable 4328260 Halogen Lamp 12V 75W 4345287 Optical 96 Well Fast Thermal Cycling Plate with Barcode code 128 20 plates 4346906 Black Optical Plate Tool 4305872 Splash free Support Base 43...

Page 43: ...ROI Calibration Plate TaqMan RNase P Instrument Verification Plate Chemistry Calibration Kits P N TaqMan RNase P Chemistry Verification Plate 7300 7500 systems 4350584 TaqMan RNase P Chemistry Fast Verification Plate 7500 Fast systems 4351979 The kit s appropriate for your system Applied Biosystems 7300 Real Time PCR System Spectral Calibration Kit containing Background Plate Pure Dye Plates FAM J...

Page 44: ...shown TaqMan Universal PCR Master Mix 2 No Amperase UNG 1 mL TaqMan Gene Expression Assay Eukaryotic 18S rRNA 0 3 mL Human Raji cDNA 25 ng µL 4352405 TaqMan Fast Reagent Starter Kit 7500 Fast systems not shown TaqMan Fast Universal PCR Master Mix 2 No Amperase UNG 1 25 mL TaqMan Gene Expression Assay Eukaryotic 18S rRNA 0 3 mL Human Raji cDNA 25 ng µL 4352407 Genomic Assays Catalog CD not shown 43...

Page 45: ...ms 7300 7500 7500 Fast Real Time PCR System Installation and Maintenance Guide this document 4378657 Applied Biosystems 7300 7500 7500 Fast Real Time PCR System Getting Started Guides Absolute Quantification 4378656 Relative Quantification 4378655 Allelic Discrimination 4378653 Plus Minus 4378652 Applied Biosystems Real Time PCR Systems Chemistry Guide 4378658 Applied Biosystems 7500 Fast Real Tim...

Page 46: ... Real Time PCR System Site Preparation Guide Refer to this guide for weights dimensions and electrical requirements Setting Up the System 1 Open the access door to the 7300 7500 7500 Fast system a Insert a thin screwdriver into the keyhole on the edge of the access door then push to unlatch the door b Open the access door 2 Verify that the heated cover assembly is pulled fully toward the front of ...

Page 47: ...e Power cords for different voltages are provided in the packing kit Connect the cord with the receptacle appropriate for your voltage then discard remaining cables 6 Press the power button at the lower right front panel then wait for the 7300 7500 7500 Fast system to boot about 30 seconds 7 When the Power status light on the lower left front panel lights press the tray to open it 8 Remove the pac...

Page 48: ...uide Notes 9 Close the tray door then press the power button again to power off the instrument Note When closing the instrument tray door apply pressure to the right side of the tray and at an angle If you have additional hardware that you want to install see page 9 do so now Do not connect the USB cable to the 7300 7500 7500 Fast instrument or computer at this time Press here Press here ...

Page 49: ... the 7300 7500 7500 Fast System Performing the ROI Calibration Performing the Background Calibration and Optical Calibration Performing the Pure Dye Calibration Verifying the Instrument Performance Before Starting the Installation Unpacking and Setting Up the Instrument See page 32 Troubleshooting See page 28 Powering On the Instrument See page 25 Installing the SDS Software for the 7300 7500 7500...

Page 50: ...tem Setup wizard starts automatically after a slight delay when you insert the CD Time Required 45 minutes Materials Required IMPORTANT If you plan to configure the 7300 7500 7500 Fast system computer with additional software see page 10 complete the installation of the 7300 7500 7500 Fast system as described in this guide before installing other software Installing third party software to the 730...

Page 51: ... from the Software and Documentation kit 2 Power on the computer then log in as a Windows operating system administrator as described in the Applied Biosystems Real Time PCR System Computer Setup Guide 3 Insert the SDS Software CD into the CD drive of the computer The Real Time System Setup wizard starts automatically after a short delay If the wizard does not automatically start double click My C...

Page 52: ... and Maintenance Guide Notes 6 In the Software Installation Materials Install the Software page click The InstallShield Wizard opens 7 In the Welcome page click 8 In the End User License Agreement page click to accept the agreement 9 In the Choose Destination Location page click to accept the default location 10 In the New Installation page click to start installing the software Click Click Click ...

Page 53: ...er the name of your business or organization c In the Registration Code field enter the registration code located on the CD case d Click 13 If a Network communications error is displayed click The installation will complete successfully even if this error is displayed Note This message is displayed if your computer is not connected to a network If you do not plan to connect this computer to a netw...

Page 54: ...c driver for the instrument which does not allow the instrument to communicate with the SDS software IMPORTANT Make sure that the computer Hibernate power setting is disabled If the Hibernate setting is enabled data collection stops when the computer goes into Hibernate mode For more information see Setting the Display Settings and Power Options on page 137 Powering On 1 Connect the Universal Seri...

Page 55: ...tor is flashing make sure the tray is closed If the red Error indicator is lit see Troubleshooting Front Panel Indicators on page 32 When the green Power indicator is lit not flashing Communication is established between the computer and the instrument The Windows XP operating system recognizes the instrument as new hardware The Windows XP operating system automatically installs the drivers needed...

Page 56: ...ce Guide Notes Performing the Instrument Function Test 1 If the SDS software is not running in the desktop double click the icon for the SDS software 2 In the SDS software click or select File New 3 In the New Document wizard click to accept the default parameters 4 If an ROI error is displayed click You perform the ROI calibration after you perform the Function test Double click Select Click ...

Page 57: ...a Click b Examine the Pass Fail column If all the function tests Pass go to step 7 If any of the function tests Fail see Troubleshooting Function Tests on page 33 c Click 7 Select File Close to close the plate document 8 When the software prompts you to save the plate document click 9 In the SDS software select File Exit Continue with Performing the Regions of Interest ROI Calibration on page 35 S...

Page 58: ...instrument door caused the error and installation continues 2 If the red Error indicator remains lit a Open the instrument door b Pull the heated cover door to verify that it is closed c Close the instrument door If the green Power On indicator lights up the open heated cover door caused the error and installation continues 3 If the red Error indicator remains lit verify that the Windows desktop i...

Page 59: ...wer off the 7300 7500 7500 Fast instrument b Wait for 30 seconds c Power on the 7300 7500 7500 Fast instrument 6 If the red Error indicator remains lit contact Applied Biosystems technical support see page xiii or your service representative Troubleshooting Function Tests Condition USB Test Failure 1 Power off the instrument wait 30 seconds then power on the instrument 2 Perform the Instrument Fun...

Page 60: ...entative Condition Lamp Test Failure 1 Power off the instrument wait 30 seconds then power on the instrument 2 Perform the Instrument Function Test see page 30 3 If the test fails again select Instrument Lamp Status Replacement 4 If the Lamp Status Replacement dialog box reports the lamp status as Failed replace the halogen lamp see Replacing the Halogen Lamp on page 118 5 Perform the Instrument F...

Page 61: ...Performing the Background Calibration and Optical Calibration Performing the Pure Dye Calibration Verifying the Instrument Performance Before Starting the Installation Unpacking and Setting Up the Instrument Installing the Software and Powering On the Instrument See page 41 Creating a Plate Document for the ROI Calibration See page 38 Preparing the ROI Calibration Plate See page 48 Troubleshooting...

Page 62: ...I Calibration Perform a regions of interest ROI calibration When installing the system you must perform all calibrations in this order ROI background optical 7500 7500 Fast systems only pure dye instrument verification Every 6 months or as often as necessary depending on instrument use After replacing the lamp IMPORTANT After every ROI calibration you must perform a background calibration optical ...

Page 63: ... ROI calibration maps the positions of the wells on the sample block so that the software can associate increases in fluorescence during a run with specific wells of the plate Because the instrument uses a set of optic filters to distinguish the fluorescence emissions gathered during runs you must generate a calibration image for each individual filter to account for minor differences in the optic...

Page 64: ...st Plates 7500 Fast Systems Notched corner at top right Vortex standard plates to ensure complete mixing then centrifuge to ensure that all reagents are contained in the bottom of the well Note Optical 96 Well Fast Plates do not fit correctly into the standard block Notched corner at top left Centrifuge Fast plates to ensure that all reagents are in the bottom of the well IMPORTANT Do not vortex F...

Page 65: ...e fluorescent dye in the wells of the plate is photosensitive Prolonged exposure to light can diminish the fluorescence signal strength of the plate 5 Remove the ROI calibration plate from its packaging Leave the optical film on the plate Do not discard the packaging for the ROI calibration plate The plate can be used up to three times if it is stored in its original packaging sleeve 1 Standard pl...

Page 66: ...plate holder in the instrument Ensure that the plate is properly aligned in the holder Load standard plates 7300 7500 system with the notched A12 position at the top right of the tray Load Fast plates 7500 Fast system with the notched A1 position at the top left of the tray 3 Close the tray Apply pressure to the right side of the tray and at an angle Continue with Creating a Plate Document for the...

Page 67: ...ecessary to name or save the plate document The SDS software automatically saves the ROI data to a set of calibration files on the computer hard drive If a Network communications error is displayed click You can perform the ROI calibration even if this error is displayed Note This message is displayed if your computer is not connected to a network If you do not plan to connect this computer to a n...

Page 68: ...he ROI Calibration 42 Applied Biosystems 7300 7500 7500 Fast Real Time PCR System Installation and Maintenance Guide Notes 3 In the SDS software select Instrument Calibrate 4 In the warning dialog box click to lower the sample block The ROI Inspector dialog box opens Select Click ROI Inspector ...

Page 69: ...alog box right click the black area of the window then select Display Properties 6 In the Image Viewer Control Properties dialog box a Select Show Saturation b In the Sat Threshold field enter 4000 c Click 7 In the ROI Inspector dialog box set the lamp control a Click b Select Idle from the Lamp Control drop down list sets the lamp to lower voltage for ROI calibration Continue with Performing the ...

Page 70: ...n oversaturated image Wells in an acceptable image Must be as bright as possible without oversaturating When you generate the ROI calibration on page 45 a warning is displayed if wells are oversaturated Can contain some but do not have to contain any red pixels which represent saturation 4 If your ROI image is acceptable skip to step 5 If your ROI image is oversaturated decrease the Exposure Time ...

Page 71: ...the ROI Inspector window If the software displays Do this a Click b Decrease the value in the Exposure Time field see step 1a by half c Repeat steps 2 and 5 A very faint ROI image or you cannot generate a successful calibration See Troubleshooting on page 48 An acceptable Calibration Image Note A green circle indicates that the region of interest for the well position is bright enough Verify that ...

Page 72: ... for all the Filters displays OK 8 Click to close the ROI Inspector 9 In the SDS software select File Close 10 When prompted to save the plate document click Unloading the Plate PHYSICAL INJURY HAZARD During instrument operation the sample block can be heated to 100 C Before performing the following procedure be sure to wait until the sample block reaches room temperature 1 In the ROI Inspector di...

Page 73: ...at room temperature in the packaging The optical calibration you perform after background calibration uses the ROI calibration plate If you be perform background and optical calibrations on another day return the packaged plate to the spectral calibration kit in the freezer Note Do not discard the ROI calibration plate If you store the plate in its packaging sleeve at 20 to 25 C you can use the pl...

Page 74: ...mage Condition The ROI image is faint or you cannot generate a successful calibration The sample block may be in its lowered position If the CCD Control Settings in the ROI Inspector displays click to raise the sample block Check that the heated cover assembly is pulled all the way forward to ensure that the tray can be pushed in properly Click GR2482 7500 7500 fast heated cover assembly GR2482 He...

Page 75: ...ifying the Instrument Performance Before Starting the Installation Performing the ROI Calibration Unpacking and Setting Up the Instrument Installing the Software and Powering On the Instrument See page 59 See page 52 See page 54 Performing the Optical Calibration for the 7500 7500 Fast System Preparing the Background Calibration Plate Creating a Plate Document for the Background Calibration See pa...

Page 76: ...st perform all calibrations in this order ROI background optical 7500 7500 Fast systems only pure dye instrument verification Monthly or as often as necessary depending on instrument use After replacing the lamp IMPORTANT 7500 7500 Fast systems only You must perform an optical calibration after every background calibration Purpose of the Background Calibration A background calibration measures the...

Page 77: ... system commonly referred to as background fluorescence Background fluorescence is a composite signal found in all spectral data This signal consists of fluorescence from several sources including Background electronic signal Contaminants in the sample block The plastic consumable plates and caps Guidelines for Calibration For a new instrument Always start with a new calibration kit Make sure the ...

Page 78: ...ting a Background Plate 2 Return the spectral calibration kit to the freezer 3 Allow the background plate to warm to room temperature at least 5 minutes 4 Remove the background plate from its packaging IMPORTANT Do not discard the packaging for the background plate The background plate can be used up to three times if it is stored in its original packaging sleeve 5 Standard plates only Vortex the ...

Page 79: ... well If not centrifuge the plate again at a higher rpm and for a longer period of time IMPORTANT Do not allow the bottom of the background plate to become dirty Fluids and other contaminants that adhere to the bottom of the plate can contaminate the sample block and cause an abnormally high background signal Continue with Creating a Plate Document for the Background Calibration on page 54 Correct...

Page 80: ...ear c Select Template Blank Document d In the Operator field enter your name e In the Comments field enter any additional information that you want to save to the file such as the plate bar code f In the Plate Name field enter Background_ date in DDMMYY format For example the name for a plate run on May 31 2005 would be Background_310505 g Click 3 In the SDS software select File Save As 4 In the S...

Page 81: ...s raised position locking the tray position To lower the block select Instrument Calibrate then exit the ROI Inspector 2 In the SDS software a Select the Instrument tab b Click The instrument begins the background calibration run Note Before starting the run the instrument may pause up to 10 minutes to allow the heated cover to reach the correct temperature Continue with Analyzing the Background C...

Page 82: ...lysis is successful Click then go to step 4 Image exposure is too low The software stopped the extraction because several raw spectra are at or below the detectable threshold for the calibration a Click b Verify that the instrument contains the background plate c Test the lamp See Monitoring Lamp Status on page 117 d Run the background plate again e If the SDS software continues to display the Ima...

Page 83: ...5 Select all wells of the plate document 6 Inspect the raw data for irregular spectral peaks that exceed the following fluorescent standard units FSU If one or more wells produce raw spectra that exceed the specified FSU the background plate or the sample block could contain a fluorescent contaminant Determine the source of the contamination See Troubleshooting on page 64 7 Select File Close a b C...

Page 84: ...ture 1 When the run is complete remove the background plate a Press the tray to open it b Remove the background plate c Press the tray to move it into the instrument 2 Place the background plate inside its packaging sleeve then return it to the spectral calibration kit in the freezer IMPORTANT Do not discard the background plate If you store the background plate in its original packaging sleeve yo...

Page 85: ...erform an optical calibration When installing the system you must perform all calibrations in this order ROI background optical 7500 7500 Fast systems only pure dye instrument verification After every 7500 7500 Fast system background calibration Preparing the ROI Calibration Plate If you kept your ROI calibration plate at room temperature after performing an ROI calibration described in Chapter 4 ...

Page 86: ... Remove the ROI calibration plate from its packaging IMPORTANT Do not discard the packaging for the ROI calibration plate The plate can be used up to three times if it is stored in its original packaging sleeve 5 Standard plates only Vortex the plate for 5 seconds Do not vortex Fast plates Remaining steps apply to both standard and Fast plates 6 Briefly centrifuge the ROI calibration plate in a ce...

Page 87: ...te Blank Document d In the Operator field enter your name e In the Comments field enter any additional information that you want to save to the file such as the plate bar code f In the Plate Name field enter Calibration_ date in DDMMYY format For example the name for a calibration performed on May 31 2005 would be Calibration_310505 g Click 3 In the SDS software select File Save As 4 In the Save A...

Page 88: ...ast Real Time PCR System Installation and Maintenance Guide Notes Performing the Optical Calibration 1 In the SDS software a Select the Instrument tab b Click The instrument begins the calibration Note Before starting the calibration the instrument may pause up to 10 minutes to allow the heated cover to reach the correct temperature 2 When the run is complete click a b Click ...

Page 89: ...e Plate PHYSICAL INJURY HAZARD During instrument operation the sample block can be heated to 100 C Before performing the following procedure be sure to wait until the sample block reaches room temperature 1 Remove the ROI calibration plate a Press the tray to open it b Remove the ROI calibration plate c Press the tray to move it into the instrument 2 Place the ROI calibration plate inside its pack...

Page 90: ...0 7500 Fast instrument Such signals may indicate that either the background or the sample block contains fluorescent contaminants Common contaminants include ink residue from permanent pens powder from disposable gloves and dust To determine the source and location of the contamination 1 In the plate document for the calibration a Select the Results tab b Select the Spectra tab 2 Select all wells ...

Page 91: ...d well must be in columns 7 12 b Select columns 7 9 The raw data from the selected wells includes the irregular peak The contaminated well must be in columns 7 9 c Select wells in row E and below in columns 7 9 The raw data from the selected wells does not include the irregular peak The contaminated well must be in the first four wells of columns 7 9 d Finally by selecting each of the wells from t...

Page 92: ...matter on the bottom of the plate or on the cover 10 If the calibration fails after you use a new background plate or decontaminate the sample block perform the following test a Press the tray to open it b Load the black plate tool from the packing kit or a plate containing a piece of black paper into the plate holder c Push the tray back into the instrument 11 Perform a background calibration see...

Page 93: ...the Installation Performing the Background Calibration and Optical Calibration Performing the ROI Calibration Unpacking and Setting Up the Instrument Installing the Software and Powering On the Instrument See page 77 See page 71 See page 73 See page 72 Analyzing the Pure Dye Calibration Data Preparing for the Calibration Preparing Plates and Performing the Pure Dye Calibration Creating a Plate Doc...

Page 94: ...ing the system you must perform all calibrations in this order ROI background optical 7500 7500 Fast systems only pure dye instrument verification Every 6 months depending on instrument use IMPORTANT You must perform a background run before every pure dye calibration Because the age and use of instrument components can affect pure spectra readings Applied Biosystems recommends performing a pure dy...

Page 95: ...h reading It determines the contribution of each fluorescent dye used in the sample by comparing the raw spectra to the pure spectra file When you save a plate document after analysis the software stores the pure spectra file with the collected fluorescence data for that experiment Dye Sets The Applied Biosystems Real Time PCR Systems use the following dye sets for calibration 7300 system FAM JOE ...

Page 96: ... 7500 Fast instrument supports the detection of custom pure dyes dyes other than those provided by Applied Biosystems Custom dyes must fluoresce within the spectral range measured by the instrument 500 to 650 nm for 7300 systems 500 to 700 nm for the 7500 7500 Fast systems To add custom pure dyes to the Pure Dye set for your instrument see Creating a Custom Pure Dye Plate on page 127 ...

Page 97: ... handle the pure dye plates 1 Retrieve the spectral calibration kit from the freezer then remove all of the pure dye plates 2 Return the spectral calibration kit to the freezer 3 Allow the pure dye plates to warm to room temperature approximately 5 minutes IMPORTANT Do not remove a pure dye plate from its packaging until you are ready to run it The fluorescent dye in the wells of each pure dye pla...

Page 98: ...Assay Pure Spectra b Select Container 96 Well Clear c Select Template Blank Document d In the Operator field enter your name e In the Comments field enter any information that you want to attach to the file such as the plate bar code f Click Note It is not necessary to name or save the pure dye plate document The SDS software automatically saves the pure dye data to a calibration file on the compu...

Page 99: ...dye to calibrate b Click c If you are prompted to disconnect the plate document click d A message prompts you to load the plate Do not click Yes or No at this time Preparing and Loading a Pure Dye Plate 1 Remove the appropriate pure dye plate from its packaging IMPORTANT Do not discard the packaging for the pure dye plate The pure dye plate can be used up to three times if it is stored in its orig...

Page 100: ...the dye selected in the Pure Spectra Calibration Manager 6 Load the plate in the instrument as described in Loading the Plate on page 40 Note When closing the instrument tray apply pressure to the right side of the tray and at an angle Performing the Pure Dye Calibration In the dialog box that prompts you to load the plate see step 1d on page 73 click then wait for the run to complete 5 minutes 00...

Page 101: ...s the run a Press the tray to open it b Remove the pure dye plate from the tray c Press the tray to move it into the instrument d Place the pure dye plate inside its packaging sleeve and return it to the spectral calibration kit in the freezer Note Do not discard the pure dye plates If you store the plates in their packaging sleeves at 20 to 25 C you can use the pure dye plates up to three times a...

Page 102: ... Calibration 76 Applied Biosystems 7300 7500 7500 Fast Real Time PCR System Installation and Maintenance Guide Notes 3 After you calibrate the instrument with all pure dyes provided in your spectral calibration kit click Continue with Analyzing the Pure Dye Calibration Data on page 77 Click ...

Page 103: ...ts remain open behind the plate document displayed by the software To complete the calibration analyze all open pure dye documents as explained below 1 From the Windows menu select the plate document to analyze 2 Select the Results tab then select the Spectra tab 3 Select all wells of the plate document by clicking the upper left corner of the plate grid 4 Click or select Analysis Extract Pure Spe...

Page 104: ...ware displays Do this Pure Spectra Extraction Complete The analysis is successful Click then go to step 5 IMPORTANT The pure dye calibration is not complete at this point Before closing the plate document you must inspect the Spectra plot as explained in steps 5 to 8 Repair Message Click then go to step 5 For information on how the software auto repairs calibration spectra see About Pure Dye Spect...

Page 105: ...ion of spectral profiles that represent the fluorescence signature of each pure dye standard Each profile consists of a set of 96 lines that correspond to the fluorescence gathered from the 96 wells of the pure dye plate The SDS software plots the resulting data for each spectral profile in a graph of fluorescence versus filter When the software extracts the calibration data from a pure dye run it...

Page 106: ...t the peak for the spectra of the pure dye occurs at the correct filter for your system 7300 system see page 80 7500 system see page 82 7500 Fast system see page 84 If the peak for the spectra of the dye occurs in the wrong filter you may have run the wrong dye plate during the calibration Repeat the procedures for only that dye Table 1 Spectra for pure dyes supported by the 7300 system Instrument...

Page 107: ...ra Applied Biosystems 7300 7500 7500 Fast Real Time PCR System Installation and Maintenance Guide 81 Notes 6 7300 System ROX D 610 nm SYBR Green A 520 nm TAMRA C 580 nm VIC B 550 nm Table 1 Spectra for pure dyes supported by the 7300 system continued Instrument Pure Dye Filter Peak nm ...

Page 108: ...ra 82 Applied Biosystems 7300 7500 7500 Fast Real Time PCR System Installation and Maintenance Guide Notes Table 2 Spectra for pure dyes supported by the 7500 system Instrument Pure Dye Filter Peak nm 7500 System FAM A 520 nm JOE B 550 nm NED C 550 nm ROX D 610 nm SYBR Green A 520 nm ...

Page 109: ...lied Biosystems 7300 7500 7500 Fast Real Time PCR System Installation and Maintenance Guide 83 Notes 6 7500 System TAMRA C 580 nm VIC B 550 nm CY3 C 580 nm CY5 E 670 nm TEXAS RED D 610 nm Table 2 Spectra for pure dyes supported by the 7500 system continued Instrument Pure Dye Filter Peak nm ...

Page 110: ... Applied Biosystems 7300 7500 7500 Fast Real Time PCR System Installation and Maintenance Guide Notes Table 3 Spectra for pure dyes supported by the 7500 Fast system Instrument Pure Dye Filter Peak nm 7500 Fast System FAM A 520 nm JOE B 550 nm NED C 550 nm ROX D 610 nm SYBR Green A 520 nm ...

Page 111: ...Biosystems 7300 7500 7500 Fast Real Time PCR System Installation and Maintenance Guide 85 Notes 6 7500 Fast System TAMRA C 580 nm VIC B 550 nm CY3 C 580 nm CY5 E 670 nm TEXAS RED D 610 nm Table 3 Spectra for pure dyes supported by the 7500 Fast system continued Instrument Pure Dye Filter Peak nm ...

Page 112: ...Chapter 6 Performing the Pure Dye Calibration Evaluating Pure Dye Calibration Spectra 86 Applied Biosystems 7300 7500 7500 Fast Real Time PCR System Installation and Maintenance Guide Notes ...

Page 113: ...forming the Background Calibration and Optical Calibration Performing the ROI Calibration Unpacking and Setting Up the Instrument Performing the Pure Dye Calibration Installing the Software and Powering On the Instrument See page 94 See page 90 See page 93 See page 92 Analyzing the TaqMan RNase P Plate Data Preparing the TaqMan RNase P Verification Plate Performing the Verification Run Creating a ...

Page 114: ...ent verification After moving the instrument to another location As needed to verify the function of the 7300 7500 7500 Fast instrument Purpose of RNase P Runs The TaqMan RNase P Instrument Verification Plate run verifies the performance of an Applied Biosystems 7300 7500 7500 Fast Real Time PCR System The RNase P plate is pre loaded with the reagents necessary for the detection and quantification...

Page 115: ...asses the verification if the analyzed data demonstrates that the instrument distinguishes between 5 000 and 10 000 genome equivalents with a 99 7 confidence level IMPORTANT Up to six outlier wells from each unknown replicate group in a 96 well TaqMan RNase P Instrument Verification Plate can be omitted from the analysis to meet specifications GR2341 GR2341 Gemini 96 well plate one sample blue 500...

Page 116: ... TaqMan RNase P Verification Plate Kit from the freezer remove the RNase P plate then allow the plate to warm to room temperature approximately 5 minutes 2 Remove the RNase P plate from its packaging Note If the RNase P plate contains a compression pad remove it from the plate Applied Biosystems does not recommend the use of compression pads with the 7300 7500 7500 Fast system 3 Standard plates on...

Page 117: ...ce Guide 91 Notes 7 5 Verify that the liquid in each well of the plate is at the bottom of the well If not centrifuge the plate again at a higher speed and for a longer period of time Continue with Creating a Plate Document for the RNase P Verification Run on page 92 Correct Incorrect Liquid is at bottom of well Not centrifuged with enough force or Not centrifuged for enough time ...

Page 118: ... Operator field enter your name e In the Comments field enter any information that you want to attach to the file such as the plate bar code f In the Default Plate Name field enter the bar code of the RNase P plate g Click 3 In the SDS software select File Save As IMPORTANT Do not modify the plate document The sample detector and method for the run are coded into the software 4 Save the document I...

Page 119: ... document a Select the Instrument tab b Click The instrument begins the run which lasts 1 5 to 2 hours using a 7300 7500 Real Time PCR system or 40 minutes using a 7500 Fast Real Time PCR system Note Before starting the run the instrument may pause up to 10 minutes to allow the heated cover to reach the correct temperature 3 When the run is complete click Continue with Analyzing the TaqMan RNase P...

Page 120: ...aqMan RNase P Plate Data 1 In the SDS software select Analysis Analysis Settings 2 In the Analysis Settings dialog box a Select Auto Ct b Click 3 In the SDS software click or select Analysis Analyze The SDS software analyzes the run data and displays the results in the Results tab 4 Access the results In the plate document a Select the Results tab b Select the Amplification Plot tab Select b a Sel...

Page 121: ... that differ significantly from the average for the associated replicate wells If included in the calculations these outlying data outliers can result in erroneous measurements 7 If outliers are present reanalyze the run a In the plate grid select the wells that produced the outlying data b Select View Well Inspector Click Outliers 1 13 25 37 49 61 73 85 2 14 26 38 50 62 74 86 3 15 27 39 51 63 75 ...

Page 122: ...epeat step 7 on the previous page for other wells with outlying data IMPORTANT Up to six wells from each replicate group in a RNase P plate can be omitted to meet specifications If the analysis contains more than six outliers troubleshoot the RNase P verification run See Troubleshooting on page 99 9 In the Results tab select the Standard Curve tab 10 In the Standard Curve tab verify that the R2 va...

Page 123: ...ate the verification value for the 10000 copy population a In the Report tab scroll to a sample in the 10K population b Apply the values in the Mean Qty and StdDev Qty columns to the following equation 10K value Mean Qty 3 StdDev Qty 14 Compare the verification values for the 10000 and 5000 copy populations If the 10K value step 13 is greater than the 5K value step 12 the 7300 7500 7500 Fast syste...

Page 124: ... Guide Notes Unloading the Plate PHYSICAL INJURY HAZARD During instrument operation the sample block can be heated to 100 C Before performing the following procedure be sure to wait until the sample block reaches room temperature 1 Remove the RNase P plate a Press the tray to open it b Remove the RNase P plate c Press the tray to move it into the instrument 2 Discard the plate ...

Page 125: ...old the plate up to a light source and verify that all wells contain the same volume of fluid If there are differences in fluid volumes check the heat seal of the wells with lower volumes for signs of damage or evaporation Also compare the position of the wells that have lower volumes with the outliers that you have removed from the plate If the well positions coincide the heat seal on the plate m...

Page 126: ...Chapter 7 Verifying the Instrument Performance Troubleshooting 100 Applied Biosystems 7300 7500 7500 Fast Real Time PCR System Installation and Maintenance Guide Notes ...

Page 127: ...ast System Before Starting the Installation Performing the Background Calibration and Optical Calibration Performing the ROI Calibration Unpacking and Setting Up the Instrument Performing the Pure Dye Calibration Verifying the Instrument Performance Installing the Software and Powering On the Instrument See page 104 Performing a Long Term Shutdown See page 102 Performing a Short Term Shutdown ...

Page 128: ...enance Guide Notes Performing a Short Term Shutdown Perform the short term shutdown procedure if you will use the instrument within 7 days Time Required 5 minutes Performing a Short term Shutdown 1 Press the tray to open it 2 If the tray contains a plate remove it then press the tray to move it into the instrument 3 Press the instrument power button Su M T W Th F S 1 week ...

Page 129: ...hort Term Shutdown Applied Biosystems 7300 7500 7500 Fast Real Time PCR System Installation and Maintenance Guide 103 Notes 8 4 Power off the computer and monitor a Select Shut Down b In the Shut Down Windows dialog box not shown select Shut Down c Power off the monitor ...

Page 130: ...step 8 on page 21 Performing a Long term Shutdown 1 Press the tray to open it 2 If the tray contains a plate remove it 3 Load the packaging plate into the tray Note If the shipping plate is not available substitute an unused reaction plate During storage the instrument optics block rests on the plate to protect the optics block 4 Press the tray to move it into the instrument 5 Press the instrument...

Page 131: ...ng System Software and Service Packs 126 Recommended Maintenance Schedule Weekly Maintenance Tasks Check disk space Archive or back up SDS plate document files see page 107 Cycle the computer and instrument power power off then power on the computer and instrument Wipe instrument surfaces with a lint free cloth IMPORTANT Never use organic solvents to clean the 7300 7500 7500 Fast system Monthly Ma...

Page 132: ...ackground calibration to check for contamination If any parts of the optics are replaced or moved you must run a background calibration an ROI calibration an optical calibration 7500 7500 Fast only and a pure dye calibration Miscellaneous Maintenance Tasks Perform the following tasks as needed to resolve problems as they arise Decontaminate the sample block see page 108 Move the 7300 7500 7500 Fas...

Page 133: ...e on the hard drive and optimizes performance if you remove old data after backing up See Choosing a Backup Storage Device on page 10 for more information about backup storage devices Developing a Data Management Strategy Applied Biosystems recommends developing a strategy for dealing with the files produced by the SDS software During a single day of real time operation the Applied Biosystems 7300...

Page 134: ...ple block can be heated to 100 C Before performing the following procedure be sure to wait until the sample block reaches room temperature The following procedure explains how to eliminate fluorescent contaminants from the sample block of the 7300 7500 7500 Fast instrument Perform the procedure to resolve problematic background runs where one or more wells consistently exhibit abnormally high sign...

Page 135: ...64 2 Remove the plate and the tray holder 3 Close the tray Apply pressure to the right side of the tray and at an angle 4 Manually raise the block from the ROI Inspector window a Click or select File New b In the New Document wizard click c In the SDS software select Instrument Calibrate d In the warning dialog box click to lower the sample block The ROI Inspector dialog box opens e In the ROI Ins...

Page 136: ... the door b Open the access door 7 Move the heated cover door to the back of the instrument 8 Clean the contaminated wells of the sample block using a small volume of deionized water a Pipette a small volume of deionized water into each contaminated well b Pipette the water up and down several times to rinse the well c Pipette the water to a waste beaker d Using a cotton swab scrub inside of each ...

Page 137: ...e on page 125 12 To confirm that you have eliminated the contamination perform a background calibration run see Performing the Background Calibration and Optical Calibration on page 49 13 If the contamination is still present repeat steps 1 through 7 then go to step 14 CHEMICAL HAZARD Ethanol is a flammable liquid and vapor Exposure causes eye skin and respiratory tract irritation and may cause ce...

Page 138: ...ntamination If the contamination is still present repeat steps 1 through 7 then go to step 16 IMPORTANT Always use deionized water to rinse wells after cleaning with bleach or ethanol solution CHEMICAL HAZARD Sodium hypochlorite bleach is a liquid disinfectant that can be corrosive to the skin and can cause skin depigmentation Read the MSDS and follow the handling instructions Wear appropriate pro...

Page 139: ...wells of the sample block and to verify that you have eliminated the contamination IMPORTANT Always use deionized water to rinse wells after cleaning with bleach or EtOH solution If contamination is present contact Applied Biosystems technical support see page xiii 18 Ensure that the heated cover assembly door is completely closed If it is not an error message is displayed 10 bleach solution ...

Page 140: ...efragment Cleaning Up the Disk 1 In the Windows desktop select All Programs 2 Select Accessories System Tools Disk Cleanup 3 Select the drive to clean up then click Click at any additional prompts 4 Repeat for remaining drives Defragmenting 1 In the Windows desktop select All Programs 2 Select Accessories System Tools Disk Defragmenter 3 At the top of the dialog box select the Volume hard drive to...

Page 141: ...7500 Fast instrument IMPORTANT Moving your Applied Biosystems 7300 7500 7500 Fast Real Time PCR System can create subtle changes in the alignment of the instrument optics 1 Load the packing plate or empty 96 well plate in the instrument 2 Manually raise the block from the ROI Inspector window a Click or select File New b In the New Document wizard click c In the SDS software select Instrument Cali...

Page 142: ... page 87 If the run passes recalibrations are not necessary If the run fails perform steps steps 6 through 9 to recalibrate the instrument 6 Perform an ROI calibration see page 35 7 Perform a Background calibration see page 49 Perform an optical calibration if you are using a 7500 or 7500 Fast system see page 59 8 Perform a pure dye calibration see page 67 9 Perform an instrument verification run ...

Page 143: ...en Lamp on page 118 Warning Messages Three warning messages can be displayed before or during a run that indicate low lamp current Message Description Warning Cannot detect sufficient current from lamp Either lamp is not installed properly or needs to be replaced Displayed at the start of a run if the lamp current has fallen below the acceptable percentage You cannot proceed with the run You must ...

Page 144: ...HYSICAL INJURY HAZARD Wear disposable powder free gloves when handling the lamp to prevent burns and to prevent shortening the life of the replacement lamp WARNING This instrument is designed for 12V 75W halogen lamps only Replace the halogen lamp after approximately 2000 hours of life Time Required 30 minutes Materials Required Replacing the Lamp IMPORTANT Wear powder free gloves when you handle ...

Page 145: ...Allow sufficient time for the lamp to cool and put on protective powder free gloves before handling it 3 Remove the lamp from the instrument a Slide the lamp release lever downward b Firmly grasp the lamp and lift it up and out of the slotted mount Do not touch the lamp without powder free gloves Finger prints shorten the lamp life 4 Inspect the lamp for signs of failure carbon typically coats the...

Page 146: ...7500 Fast Real Time PCR System Installation and Maintenance Guide Notes 6 Close the access door 7 Plug in and power on the 7300 7500 7500 Fast system 8 In the SDS software select Instrument Calibrate 9 In the ROI Inspector dialog box select Lamp Control Idle Select Select ...

Page 147: ...illuminated the replacement halogen lamp may be defective Replace the lamp again If the second lamp does not illuminate check the instrument fuses for failure see page 122 11 Perform the calibrations listed below after replacing the lamp See Chapter 4 Performing the Regions of Interest ROI Calibration Chapter 5 Performing the Background Calibration and Optical Calibration Chapter 6 Performing the ...

Page 148: ...against the risk of fire replace fuses only with listed and certified fuses of the same type and rating as those currently in the instrument Time Required 30 minutes Materials Required Replacing the Fuses 1 Turn off the instrument then unplug it 2 Using a flat head screwdriver unscrew and remove the fuse holders from the instrument Powder free gloves Flathead screwdriver Fuses 2 12 5A 250V 5 x 20 ...

Page 149: ... Replace failed fuses with a 12 5A 250V 5 x 20 mm fuse Note The voltage and amperage ratings are on the fuse holder 5 Replace the fuse holder into the instrument 6 Plug in then power on the instrument The installation is successful if the instrument powers on Note Fuse failure can result from fluctuations in the supplied power to the instrument To prevent further failures consider installing an el...

Page 150: ...systems representative New versions of the Microsoft Windows operating system can conflict with the SDS software and make the instrument inoperable Service Pack Updates To install a service pack to update the operating system review the release notes provided with the SDS software installation for compatibility issues Note Applied Biosystems service engineers maintain the operating system software...

Page 151: ...ear powder free gloves while creating the background plate 1 Remove an Applied Biosystems 96 Well Optical Reaction Plate or 96 Well Fast Reaction Plate from its box and place it on a clean dry surface 2 Aliquot 50 µL 7300 7500 system or 20 µL 7500 Fast system of deionized water to each well of the reaction plate 3 Seal the plate using an optical adhesive cover or optical flat caps Use the plate fo...

Page 152: ...Appendix B Creating a Background Plate 126 Applied Biosystems 7300 7500 7500 Fast Real Time PCR System Installation and Maintenance Guide Notes ...

Page 153: ...r 7300 systems 500 to 700 nm for the 7500 7500 Fast systems Before using custom dyes with the 7300 7500 7500 Fast instrument you must Determine optimum dye concentration Create a custom pure dye plate Add the custom dye to the software Perform a pure dye calibration see Chapter 6 Performing the Pure Dye Calibration Materials Required Optical Adhesive Cover or Optical Flat Caps ABI PRISM Optical 96...

Page 154: ... µL volumes for the 7500 Fast system 2 Seal the wells of the reaction plate using an optical adhesive cover 3 In the SDS software click or select File New 4 In the New Document dialog box click Note It is not necessary to configure detector sample and method information for the plate document The purpose of the run is to establish the correct working concentration for the dye by viewing the intens...

Page 155: ...he block 8 In the ROI Inspector create the ROI image for each filter beginning with Filter A a In the Exposure Time field enter 1024 b Click c Select Filter A d Click e Check the image for saturation f Record the coordinate of the well that displays the brightest possible signal without saturation This well contains the best concentration of the custom pure dye for Filter A Select Click a c b No S...

Page 156: ... following procedure be sure to wait until the sample block reaches room temperature 1 In the ROI Inspector click 2 Remove the plate from the instrument a Press the tray to open it b Remove the plate c Press the tray to move it into the instrument Note If you cannot open the tray the sample block may be in its raised position locking the tray position To lower the block select Instrument Calibrate...

Page 157: ...ide 131 Notes C Adding the Custom Dye to the Software 1 In the SDS software select Tools Dye Manager 2 In the Dye Manager dialog box click 3 In the Add Dye dialog box enter a name for the custom dye then click 4 Click Run the custom pure dye plate see Chapter 6 Performing the Pure Dye Calibration Select Click Enter a name for the dye Click ...

Page 158: ...Appendix C Adding the Custom Dye to the Software 132 Applied Biosystems 7300 7500 7500 Fast Real Time PCR System Installation and Maintenance Guide Notes ...

Page 159: ...is appendix duplicates the information in the Applied Biosystems Real Time System Computer Setup Guide Unpacking the Computer and Setting Voltage 136 Connecting Components and Powering Up 138 Setting the Display Settings and Power Options 139 Connecting to the Network and Downloading Adobe Acrobat 141 Setting Up the Computer ...

Page 160: ...1 Unpack the computer boxes 2 Place the monitor computer keyboard and mouse on the bench as shown Do not connect components or the USB cable to the instrument or computer at this time Doing so may result in installation of incorrect drivers GRXXXX 7300 7500 Unpack Computer Tilt box on side then slide computer out Tower Computer Laptop Computer num lock caps lock scroll lock F12 F11 F10 F9 F8 F7 F6...

Page 161: ...nternational voltage kit PN603615 provided in the instrument packing kit Select the appropriate power cord you can discard remaining power cords IMPORTANT Outside the US use the power cord from the international voltage kit not the power cord provided in the computer carton 4 Laptop computers Connect the power cord laptop computer AC adaptors contain an auto sensing voltage detector and automatica...

Page 162: ...efer to the diagram below to verify connections 2 Power on the computer and monitor tower computer shown at right The power switch on a laptop is at the top center of the keyboard Wait for the computer to boot 3 Log on using an account with windows administrator privileges see your system administrator for more information Tower Computer Laptop Computer Monitor Power Power Keyboard Mouse If two co...

Page 163: ... Windows operating system desktop is displayed Setting the Display Settings and Power Options IMPORTANT You must have administrator rights to set the display and power options for the computer 1 Select Control Panel 2 In the top left of the Control Panel click Switch to Classic View 3 In the Control Panel window double click Display Windows desktop Click Double click ...

Page 164: ...ay Properties dialog box select the font a Click the Appearance tab b Select Font size Normal 6 In the Display Properties dialog box select color quality a Click the Settings tab b In the Screen resolution box select 1280 by 1024 pixels using the slider Note Some laptop models have a maximum resolution setting of 1024 by 768 pixels c Select Color quality Highest 32 bit 7 Click 8 If you are using a...

Page 165: ...r Turn off monitor select Never c For Turn off hard disks select Never d For System standby select Never e For System Hibernates select Never this selection on the Power Schemes tab is not displayed on laptop computers f Select the Hibernate tab not shown then ensure that the Enable Hibernation checkbox is not checked g Click IMPORTANT Make sure that the computer Hibernate power setting is disable...

Page 166: ...etwork Use of a wireless network can interfere with data collection and may result in data loss 2 Download Adobe Acrobat Reader free shareware from www adobe com Note that you must download Adobe Acrobat Reader to view document links referenced in the Real Time System Setup wizard and the Sequence Detection Systems Online Help 3 After the computer is set up proceed with the installation by using e...

Page 167: ...eparing 52 running 55 backing up SDS files 107 backup storage devices 10 biohazardous waste handling xxiii biological hazard guidelines xxiv C calibration background 50 optical 7500 7500 Fast systems only 59 pure dye 67 ROI 37 CAUTION description xvi checking shipped materials 14 chemical safety xxi xxii chemical waste safety xxii chemistry kits shipped with the instrument 17 cleaning the sample b...

Page 168: ... safety xxii H halogen bulb replacing 117 warning hot 118 119 hardware selecting additional 9 hazard icons See safety symbols on instruments hazard symbols See safety symbols on instruments hazards See safety heated cover assembly 20 I IMPORTANT description xvi indicator lights location 29 troubleshooting 29 Information Development department contacting xii Installation and Maintenance Guide overv...

Page 169: ...tion 7500 7500 Fast systems only 59 pure dye run 71 RNase P run 90 ROI calibration 38 physical hazard safety xxiv planning the installation 5 plate document 7500 7500 Fast optical calibration 61 background 54 pure dye calibration 72 RNase P plate 92 plates Fast description 38 Fast do not vortex 38 loading 40 location of notched corner 40 number of reuses 52 standard description 38 unloading 46 pow...

Page 170: ...late 73 RNase P plate 93 ROI plate 44 S safety before operating the instrument xx biological hazards xxiv chemical xxi chemical waste xxii conventions xvi electrical xxiii ergonomic xxv guidelines xxii xxiii instrument operation xx moving and lifting instrument xx moving parts xxiv moving lifting xx physical hazard xxiv repetitive motion xxv standards xxv workstation xxv safety labels on instrumen...

Page 171: ...ation data 64 cannot extract background data 64 CCD failure 34 Filter Wheel failure 34 function test 33 indicator lights 29 lamp failure 34 lamp status 117 more than 6 outliers in RNase P data 99 optical calibration 64 powering on 32 red Error indicator is lit 32 RNase P run failed 99 ROI image is faint 48 Shutter failure 34 Thermal Cycler failure 34 USB failure 33 U uncrating the instrument 12 un...

Page 172: ...146 Applied Biosystems 7300 7500 7500 Fast Real Time PCR System Installation and Maintenance Guide Index ...

Page 173: ......

Page 174: ...cal office or refer to our Web site at www appliedbiosystems com Applera is committed to providing the world s leading technology and information for life scientists Applera Corporation consists of the Applied Biosystems and Celera Genomics businesses Headquarters 850 Lincoln Centre Drive Foster City CA 94404 USA Phone 1 650 638 5800 Toll Free In North America 1 800 345 5224 Fax 1 650 638 5884 www...

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